The present invention relates to bis-benzimidazoles and related compounds, many of which are novel; to uses and compositions of such compounds; and to methods for their preparation.
The minor groove of duplex DNA is the site of non-covalent interaction of a large number of anticancer drugs, antibiotics and antiviral agents, which are believed to exert their action by competing with transcription factors or architectural proteins, such as E2F, TATA-box binding proteins or DNA topoisomerase I/II. The molecular basis of DNA recognition for a number of drugs in this super-family (notably distamycin, netropsin, berenil, pentamidine and Hoechst 33258) has been extensively studied, notably by crystallography, NMR and footprinting methods. These studies have shown that their AT preferences are a consequence of, in particular, (i) the sequence-dependent narrow width of the minor groove of B-form DNA, resulting in stabilisation by van der Waals interactions with the walls of the groove, and (ii) their ability to form specific hydrogen bonds with donor and acceptor atoms on the minor-groove edge of A:T base pairs. These factors have been utilised in the design of molecules with altered and extended recognition properties, some of which are capable of sequence-specific gene regulation. Hoechst 33258 is a bis-benzimidazole derivative (I): 
The benzene ring of one benzimidazole system is linked to the imidazole ring of the other. Such structures may be termed xe2x80x9chead to tailxe2x80x9d.
Crystallographic analyses of a number of oligonucleotide complexes with Hoechst 33258, and several of its derivatives, including a tris-benzimidazole with an extended recognition site, have shown that each benzimidazole subunit interacts with two A:T base pairs by means of a pair of cross-strand hydrogen bonds. The head-to-tail arrangement forces the site for each successive subunit to overlap the previous one by one base-pair, so that each benzimidazole group in Hoechst 33258 and other head-to-tail analogues effectively recognises 1.5 A:T base pairs.
(N Spink, D G Brown, J V Skelly and S Neidle, Nucleic Acids Res., 1994, 22, 1607; G R Clark, D W Boykin, A Czarny and S Neidle, Nucleic Acids Res., 1997, 25, 1510; G R Clark, E J Gray, S Neidle, Y-H Li and W Leupin, Biochemistry, 1996, 35, 13745).
A symmetrical head-to-head benzimidazole has recently been reported (E V Bichenkova et al, Nucleosides and Nucleotides, 1998, 17, 1651). This is 2,2xe2x80x2-bis(4-hydroxyphenyl)-6,6xe2x80x2-bis-benzimidazole. It was prepared by the reaction of 3,3xe2x80x2-diaminobenzidine with ethyl 4-hydrozybenzimidate hydrochloride in boiling acetic acid. It is reported that this compound binds specifically to t-RNA, as do Hoechst 33258 and distamycin.
Molecular modelling, based on these structural studies, suggested to us that compounds with a symmetric head-to-head benzimidazole arrangement, and other analogues, could also bind in the minor groove in an effective manner. This arrangement would extend the size of the bis-benzimidazole recognition site from three (in Hoechst 33258 and analogues) to four consecutive A:T base pairs, with distinctive cross-strand hydrogen bonding involving each base pair. This arrangement thus extends the effective recognition of each benzimidazole sub-unit to two A:T base pairs. The modelling used the structures of the self-complementary duplex sequences d(CGCGAATTCGCG) and d(CGCAAATTTGCG), as found in several relevant drug complexes. It suggested that the central 5xe2x80x2-AATT sequence would be an optimal site for the head-to-head motif and for maintenance of helical register with all four consecutive base pairs without necessitating significant DNA conformational change. We have also appreciated that the motif could provide a platform for adding further functionality which could specifically recognise bases beyond the central core of four A:T base pairs.
In a first aspect the present invention provides a compound of the formula II (and tautomers): 
where X1 and X2 are each independently selected from NH, O and S (X1 and X2 preferably being the same) and A1 and A2 are independently selected from: xe2x80x94Yxe2x80x94Qxe2x80x94Z where Y is selected from O, NH and S; Q is selected from xe2x80x94(CH2)nxe2x80x94 where n is 0-10 and carbocyclic 3-7 membered rings; and Z is selected from H, NR3R4 (where each of R3 and R4 is selected from H; a saturated or unsaturated carbon chain of up to 10 carbon atoms or a ring of up to 7 carbon atoms), or a heterocyclic ring preferably of 3-8 atoms with 1-3 heteroatoms selected from O, S and N; and xe2x80x94LM where M is an alkylating agent functionality, particularly a nitrogen mustard functionality e.g. xe2x80x94N(CH2CH2Cl)2, preferably Aryl-N(CH2CH2Cl)2, and L is a linking group e.g. 
wherein A1 and A2 are preferably the same; or a pharmaceutically acceptable salt thereof; with the proviso that if X1=X2=NH, then A1 and A2 are not both OH.
In a second aspect the invention provides a pharmaceutical composition comprising a compound of the formula II where X1 and X2 are each independently selected from NH, O and S (X1 and X2 preferably being the same) and A1 and A2 are independently selected from: xe2x80x94Yxe2x80x94Qxe2x80x94Z where Y is selected from O, NH and S; Q is selected from xe2x80x94(CH2)nxe2x80x94 where n is 0-10 and carbocyclic 3-7 membered rings; and Z is selected from H, NR3R4 (where each of R3 and R4 is selected from H; a saturated or unsaturated carbon chain of up to 10 carbon atoms or a ring of up to 7 carbon atoms), or a heterocyclic ring preferably of 3-8 atoms with 1-3 heteroatoms selected from O, S and N; and xe2x80x94LM where M is an alkylating agent functionality, particularly a nitrogen mustard functionality e.g. xe2x80x94N(CH2CH2Cl)2, preferably Aryl-N(CH2CH2Cl)2, and L is a linking group e.g. 
wherein A1 and A2 are preferably the same; or a pharmaceutically acceptable salt thereof.
In a third aspect the invention provides the use of a compound of formula (II) as defined in the second aspect in the manufacture of a composition for therapeutic use as a cytotoxic, antibiotic or antiviral agent.
A fourth aspect of the present invention is the use of a compound as described in the first aspect of the invention, disregarding the proviso, in a method of therapy. Conditions which may be treated include gene-based diseases, including, for example, neoplastic diseases, and also bacterial, parasitic and viral infections. Any condition which may be treated by the regulation of gene expression may be treated using compounds of the invention. In accordance with this aspect of the present invention, the compounds provided may be administered to individuals. Administration is preferably in a xe2x80x9ctherapeutically effective amountxe2x80x9d, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula II, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site .of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-freeand has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Preferred compounds of the invention are symmetrical: that is both X1=X2 and A1=A2.
In a further aspect the invention provides a method of preparing a symmetrical compound of formula (II) by condensing a biphenyl compound of formula (III) 
with a benzaldehyde of formula (IV) 
Thus a preferred synthetic route to symmetrical bis-benzimidazoles (V) is as shown in the accompanying FIG. 1.